I want to do mice splenocyte/lymphocyte culture in presence specific antigenand the measure the response lymphocyte by MTT and cytocine. can you give me a protocole that explain (Which kind of medium are you using (RPMI-SVF or Cerotini)? Which cell concentration? Which culture plate (24well, 96well, etc.)? What incubation time? The read-out should be supernatant analysis for cytokine production and proliferation assay using MTT. Additional question: are you adding bolus of your stimulating agent at different time-points after the beginning or just one shot? What is your best splenocyte/lymphocyte restimulation protocol?
I hope this article helps Hamidreza:
In vitro stimulation of Balb/c and C57 BL/6 splenocytes by a recombinantly produced banana lectin isoform results in both a proliferation of T cells and an increased secretion of interferon-gamma, International Immunopharmacology, Volume 10, Issue 1, January 2010, Pages 120–129.
Andrew :-)
What kind of lymphocytes?
Are you looking to activate naive lymphocytes/splenocytes? Or measure response from antigen-experienced lymphocytes?
If you want to study recall responses of antigen-primed T-cells in each of these tissues, I would imagine that you could probably look at common ELISPOT protocols in terms of well format, incubation time, etc. and easily adapt them to an MTT read out.
I want to measure response from antigen-experienced lymphocytes by MTT test
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